Downregulation of Max dimerization protein 3 is involved in decreased visceral adipose tissue by inhibiting adipocyte differentiation in zebrafish and mice.

BACKGROUND: The diet-induced obesity model of zebrafish (DIO-zebrafish) share a common pathophysiological pathway with mammalian obesity. OBJECTIVES: We aimed to investigate the role of Max dimerization protein 3 (MXD3) in visceral fat accumulation and adipocyte differentiation, by conducting knockdown experiments using zebrafish and mouse preadipocytes. METHODS: To identify genes related to visceral adiposity, we conducted transcriptome analyses of human and zebrafish obese populations using the Gene Expression Omnibus and DNA microarray. We then intraperitoneally injected morpholino antisense oligonucleotides (MO-mxd3) to knockdown mxd3 gene expression in DIO-zebrafish and measured several parameters, which reflected human obesity and associated metabolic diseases. Finally, lentiviral Mxd3 shRNA knockdown in mouse 3T3-L1 preadipocytes was conducted. Quantitative PCR analyses of several differentiation markers were conducted during these gene knockdown experiments. RESULTS: We found that MXD3 expression was increased in the obese population in humans and zebrafish. Intraperitoneal MO-mxd3 administration to DIO-zebrafish suppressed the increase in body weight, visceral fat accumulation and the size of mature adipocytes. Subsequently, dyslipidemia and liver steatosis were also ameliorated by MO-mxd3. In mouse adipocytes, Mxd3 expression was drastically increased in the early differentiation stage. Mxd3 shRNA inhibited preadipocyte proliferation and adipocyte maturation. Quantitative PCR analyses showed that the early differentiation marker, CCAAT/enhancer-binding protein delta (Cebpd) and late differentiation markers (CCAAT/enhancer-binding protein, alpha and peroxisome proliferator-activated receptor gamma) were downregulated by Mxd3 knockdown in 3T3-L1 cells and DIO-zebrafish. Subsequently, mature adipocyte markers (adiponectin and caveolin 1 for zebrafish, and fatty acid binding protein 4 and stearoyl-coenzyme A desaturase 1 for mouse adipocytes) were also decreased. CONCLUSION: Mxd3 regulates preadipocyte proliferation and early adipocyte differentiation via Cebpd downregulation in vitro and in vivo. Integrated analysis of human and zebrafish transcriptomes allows identification of a novel therapeutic target against human obesity and further associated metabolic disease.

X-ray structure of T4 endonuclease V: an excision repair enzyme specific for a pyrimidine dimer.

The x-ray structure of T4 endonuclease V, an enzyme responsible for the first step of a pyrimidine-dimer-specific excision-repair pathway, was determined at a 1.6-angstrom resolution. The enzyme consists of a single compact domain classified into an all-alpha structure. This single domain has two distinct catalytic activities; it functions as a pyrimidine dimer glycosylase and as an apurinic-apyrimidinic endonuclease. The amino-terminal segment penetrates between two major helices and prevents their direct contact. The refined structure suggests the residues involved in the substrate binding and the catalysis of the glycosylation reaction.

Functional analyses of the domain structure in the Holliday junction binding protein RuvA.

BACKGROUND: Homologous recombination is crucial for genetic diversity and repairing damaged chromosomes. In Escherichia coli cells, the RuvA, RuvB and RuvC proteins participate in the processing of an important intermediate, the Holliday junction. The RuvA-RuvB protein complex facilitates branch migration of the junction, depending on ATP hydrolysis. The atomic structure of RuvA should enable critical questions to be addressed about its specific interactions with the Holliday junction and the RuvB protein. RESULTS: The crystal structure of RuvA shows the tetrameric molecules with a fourfold axis at the center. Each subunit consists of three distinct domains, some of which contain important secondary structure elements for DNA binding. Together with the detailed structural information, the biochemical assays of various mutant RuvA proteins and domains, isolated by partial proteolysis, allowed us to define the functional roles of these domains in Holliday junction binding and the RuvB interaction. CONCLUSIONS: The RuvA molecule is formed by four identical subunits, each with three domains, I, II and III. The locations of the putative DNA-binding motifs define an interface between the DNA and the Holliday junction. Domain III is weakly attached to the core region, comprising domains I and II; the core domains can form a tetramer in the absence of domain III. Functional analyses of the mutant proteins and the partial digestion products, including Holliday junction binding and branch-migration assays, revealed that domain III and the preceding loop are crucial for RuvB binding and branch migration, although this region is not required for the junction-DNA binding.

Crystal structure of an archaeal intein-encoded homing endonuclease PI-PfuI.

Inteins possess two different enzymatic activities, self-catalyzed protein splicing and site-specific DNA cleavage. These endonucleases, which are classified as part of the homing endonuclease family, initiate the mobility of their genetic elements into homologous alleles. They recognize long asymmetric nucleotide sequences and cleave both DNA strands in a monomer form. We present here the 2.1 A crystal structure of the archaeal PI-PfuI intein from Pyroccocus furiosus. The structure reveals a unique domain, designated here as the Stirrup domain, which is inserted between the Hint domain and an endonuclease domain. The horseshoe-shaped Hint domain contains a catalytic center for protein splicing, which involves both N and C-terminal residues. The endonuclease domain, which is inserted into the Hint domain, consists of two copies of substructure related by an internal pseudo 2-fold axis. In contrast with the I-CreI homing endonuclease, PI-PfuI possibly has two asymmetric catalytic sites at the center of a putative DNA-binding cleft formed by a pair of four-stranded beta-sheets. DNase I footprinting experiments showed that PI-PfuI covers more than 30 bp of the substrate asymmetrically across the cleavage site. A docking model of the DNA-enzyme complex suggests that the endonuclease domain covers the 20 bp DNA duplex encompassing the cleavage site, whereas the Stirrup domain could make an additional contact with another upstream 10 bp region. For the double-strand break, the two strands in the DNA duplex were cleaved by PI-PfuI with different efficiencies. We suggest that the cleavage of each strand is catalyzed by each of the two non-equivalent active sites.

Modulation of RuvB function by the mobile domain III of the Holliday junction recognition protein RuvA.

In prokaryotes, RuvA-RuvB complexes play a crucial role in the migration of the Holliday junction, which is a key intermediate of homologous recombination. RuvA binds to the Holliday junction and enhances the ATPase activity of RuvB required for branch migration. RuvA adopts a unique domain structure, which assembles into a tetrameric molecule. The previous mutational and proteolytic analyses suggested that mutations in a carboxyl-terminal domain (domain III) impair binding of RuvA to RuvB. In order to clarify the functional role of each domain in vitro, we established the recombinant expression systems, which allow us to analyze structural and biochemical properties of each domain separately. A small-angle X-ray scattering solution study, combined with X-ray crystallographic analyses, was applied to the tetrameric full-length RuvA and its tetrameric NH2 region (domains I and II) lacking the domain III. These results demonstrated that domain III can be completely separate from the tetrameric major core of the NH2 region and freely mobile in solution, through a remarkably flexible loop. Biochemical analyses indicated that domain III not only interacts with RuvB, but also modulates its ATPase activity. This modulation may facilitate the dynamic coupling between RuvA and RuvB during branch migration.

Preliminary crystallographic study of Escherichia coli RuvC protein. An endonuclease specific for Holliday junctions.

Single crystals of the RuvC protein, an Escherichia coli endonuclease specific for Holliday junctions, were grown by the microdialysis method. The crystals belong to the space group P2(1), with unit cell dimensions a = 72.8 A, b = 139.6 A, c = 32.4 A and beta = 93.0 degrees, and contain four molecules in an asymmetric unit. Diffraction data to a Bragg spacing of 2.5 A resolution has been obtained using a synchrotron X-ray source.

Crystal structure of a pyrimidine dimer-specific excision repair enzyme from bacteriophage T4: refinement at 1.45 A and X-ray analysis of the three active site mutants.

Crystallographic study of bacteriophage T4 endonuclease V, which is involved in the initial step of the pyrimidine dimer-specific excision repair pathway, has been carried out with respect to the wild-type and three different mutant enzymes. This enzyme catalyzes the cleavage of the N-glycosyl bond at the 5'-side of the pyrimidine dimer, and subsequently incises the phosphodiester bond at the apyrimidinic site through a beta-elimination reaction. The structure of the wild-type enzyme refined at 1.45 A resolution reveals the detailed molecular architecture. The enzyme is composed of a single compact domain classified as an all-alpha structure. The molecule is stabilized mainly by three hydrophobic cores, two of which include many aromatic side-chain interactions. The structure has a unique folding motif, where the amino-terminal segment penetrates between two major alpha-helices and prevents their direct contact, and it is incompatible with the close-packing category of helices for protein folding. The concave surface, covered with many positive charges, implies an interface for DNA binding. The glycosylase catalytic center, which comprises Glu23 and the surrounding basic residues Arg3, Arg22 and Arg26, lie in this basic surface. The crystal structures of the three active-site mutants, in which Glu23 was replaced by Gln(E23Q) and Asp (E23D), respectively, and Arg3 by Gln (R3Q), have been determined at atomic resolution. The backbone structures of the E23Q and R3Q mutants were almost identical with that of the wild-type, while the E23D mutation induces a small, but significant, change in the backbone structure, such as an increase of the central kink of the H1 helix at Pro25. In the catalytic center of the glycosylase, however, these three mutations do not generate notable movements of protein atoms, except for significant shifts of some bound water molecules. Thus, the structural differences between the wild-type and each mutant are confined to the remarkably small region around their replaced chemical groups. Combined with the biochemical studies and the difference circular dichroism measurements, these results allow us to conclude that the negatively charged carboxyl group of Glu23 is essential for the cleavage of the N-glycosyl bond, and that the positively charged guanidino group of Arg3 is crucial to bind the substrate, a DNA duplex containing a pyrimidine dimer. The amino terminal alpha-amino group is located at a position approximately 4.4 A away from the carboxyl group of Glu23. These structural features are generally consistent with the reaction scheme proposed by Dodson and co-workers.

Histamine is an antagonist of the acetylcholine receptor at the frog endplate.

The effects of histamine on the acetylcholine (ACh) receptor-channel complex were examined by means of voltage-clamp at the frog endplate. ACh was ionophoretically applied to the endplate. Histamine was added to the perfusate. Histamine (100 nM - 1 mM) reversibly depressed the peak amplitude of the ACh-induced inward current in a dose-dependent manner. The double reciprocal plot of the dose-response relationship between the peak ACh current and the amount of ACh applied suggested that histamine (100 microM) depressed the ACh-induced current in a competitive manner. Histamine prevented the specific ACh binding site within the receptor-channel complex from binding erabutoxin, a sea-snake venom, which binds irreversibly to the specific ACh binding site. Histamine had no detectable effects on the equilibrium potential of the endplate current but shortened the half-decay time of the endplate current in a voltage-dependent manner. It was therefore concluded that histamine blocks not only the specific ACh binding site but also interacts with the ACh-channel site. The present experiments strongly suggest that histamine can act as an antagonist to modulate nicotinic cholinergic transmission.

Glucocorticoid modulates the sensitivity of the GABAA receptor on primary afferent neurons of bullfrogs.

With intracellular and voltage-clamp recording techniques, we have demonstrated that the glucocorticoids, prednisolone and hydrocortisone at a concentration of 5 microM to 1 mM, reversibly depressed gamma-aminobutyric acid (GABA)-induced responses on primary afferent neurons of bullfrogs. An analysis with dose-response curves revealed that the glucocorticoids decreased the sensitivity of the GABAA receptor in a non-competitive manner. We suggest that glucocorticoids act as an antagonist of the GABAA receptor on primary afferent neurons, probably by reducing the number of functional GABAA receptor ionic channel complexes.

Voltage-clamp studies of the inhibition of gamma-aminobutyric acid response by glucocorticoids in bullfrog primary afferent neurons.

Acute effects of glucocorticoids on the response to gamma-aminobutyric acid (GABA) were examined in primary afferent neurons in bullfrog spinal ganglia, using intracellular and voltage-clamp recording techniques. Prednisolone and hydrocortisone (5 microM to 1 mM) caused a dose-dependent decrease in the amplitude of GABA-induced depolarization, while having no effect on the membrane potential and resistance of the neuron. Prednisolone depressed the muscimol-induced depolarization. Nipecotic acid, a blocker of GABA uptake, did not influence the inhibitory action of prednisolone. Voltage-clamp analyses showed that the inward current induced by an iontophoretic application of GABA (GABA current) was suppressed by prednisolone and hydrocortisone. The depression of the GABA current is neither due to a blockage of open channels nor a facilitation of the desensitization of GABA receptors. Prednisolone shifted the dose-response curve of the GABA current downward. The double-reciprocal (Lineweaver-Burk) plot showed that the maximum GABA current was reduced by prednisolone, suggesting a non-competitive antagonism. These results suggest that glucocorticoids suppress the GABA-induced chloride current, decreasing the number of functional channels associated with GABAA receptor.

Histamine as an endogenous antagonist of nicotinic ACh-receptor.

The mechanism of an inhibitory effect of histamine on the sensitivity of frog skeletal muscle endplate was analyzed by studying the dose-response relation between the quantity of ACh applied iontophoretically and the ACh-induced postsynaptic current (ACh current), and also the interaction between histamine and erabutoxin-b (ETX-b). The results obtained show that histamine, like curare, decreased the sensitivity of ACh-receptor in a competitive manner.

Mianserin blocks alpha 2 adrenoceptors in submucous neurones of the guinea-pig caecum.

Intracellular recordings were made from submucous plexus neurones of the guinea-pig caecum in vitro. The peak amplitude of the adrenergic inhibitory postsynaptic potential (IPSP) was depressed by mianserin in a dose-dependent manner (300 nM-100 microM). This was due to a direct blockade of postsynaptic alpha 2 adrenoceptors. The nicotinic excitatory postsynaptic potential (EPSP) and the non-cholinergic EPSP were not affected by mianserin (100 microM). The presynaptic inhibition of the release of acetylcholine, mediated by presynaptic alpha 2 receptors, was also blocked by mianserin (30 microM). The results suggest that mianserin antagonizes both pre- and post-synaptic alpha 2 adrenoceptors in enteric plexus neurones.

Presynaptic effects of cholecystokinin octapeptide on neuromuscular transmission in the frog.

Intracellular recordings were obtained from the frog sartorius muscle end-plate to investigate the effects of cholecystokinin octapeptide (CCK-8) on cholinergic transmission at the neuromuscular junction. A brief bath-application of CCK-8 (1 microM) produced a depression, followed by a long-lasting facilitation, of the amplitude and the quantal content of the end-plate potential (epp). CCK-8 had a biphasic effect, an initial depression followed by an augmentation of the frequency of the miniature epps. CCK-8 did not affect the sensitivity of the nicotinic receptor at the end-plate. These results suggest a significant role for CCK-8 in cholinergic transmission, possibly as a modulator of the evoked release of acetylcholine from motor nerve terminals.

Experimental external fixation combined with percutaneous discectomy in the management of scoliosis.

STUDY DESIGN: An assessment of the value of external fixation with or without percutaneous discectomy for the management of scoliosis in young rabbits with induced progressive thoracic scoliosis. OBJECTIVES: To investigate in an experimental setting the effect of external fixation with or without percutaneous discectomy for the management of scoliosis, as a preliminary study to precede clinical consideration. SUMMARY OF BACKGROUND DATA: External fixation of the spine using percutaneous transpedicular screws has been used clinically for cases of traumatic spinal injury, infectious spine, or chronic low back pain caused by a disc lesion. Percutaneous discectomy for the management of scoliosis has been reported. METHODS: Thirty-two young rabbits underwent partial resection of the right lower ribs. Nine rabbits were not treated after production of scoliosis and were followed as controls. At 4-6 weeks after production of scoliosis, in 23 animals, Kirschner wires were inserted percutaneously into the T9-T10 and L1-L2 disc space, and both ends were attached to an external fixator after correction of the scoliosis. In 8 of those 23 animals, percutaneous discectomy was also performed at the apex of the caudal compensatory curvature. RESULTS: In these 23 animals, the initial correction by fixation on of the caudal vertebrae was accompanied by a derotation in the apex. Five animals treated with external fixation only and four treated with combined percutaneous discectomy survived with external fixation until the age of 17 weeks and were followed to the natural cessation of the curve progression, at which the fixation was removed and a final assessment was made. The mean progression of curvature was 15.8 degrees in the group of five animals with external fixation only, and 33.8 degrees in the controls. In the group of four animals treated with supplementary percutaneous discectomy, however, the treated disc space became rigid, and the mean progression of curvature after removal of the fixation was only 5.3 degrees. CONCLUSIONS: The results of the current study suggest the potential for external fixation to allow for derotation and, when combined with percutaneous discectomy, to offer a feasible method of managing scoliosis in the human adolescent. This study was a preliminary experimental study; further experimental studies are planned to develop this novel technique.

Percutaneous suction aspiration and drainage for pyogenic spondylitis.

STUDY DESIGN: Retrospective evaluation of results in 23 cases of early-stage pyogenic spondylitis treated with percutaneous suction aspiration and drainage. OBJECTIVES: To evaluate the efficacy of percutaneous suction aspiration and drainage as a treatment method for early-stage pyogenic spondylitis. SUMMARY OF BACKGROUND DATA: Traditional surgical treatment for pyogenic spondylitis has the disadvantage of increased morbidity caused by the extensive exposure required in the presence of infection. Recently, a few case reports have described minimally invasive treatment for pyogenic spondylitis in which percutaneous suction aspiration was used. However, the efficacy of this new treatment has not yet been evaluated. METHODS: All charts, radiographs, and bacteriologic and histologic findings were reviewed. All 23 patients who received the new treatment were observed clinically and radiographically, to evaluate the efficacy of the treatment. RESULTS: To date, all patients have been observed for more than 2 years. Twenty (87%) of the 23 patients have shown good results according to the evaluation. The causative organism was identified using tissue culturing in 12 (52%) of the 23 patients. The causative organism was Staphylococcus aureus in 8, and Staphylococcus epidermidis, Candida albicans, Pseudomonas aeruginosa, and Propionibacterium acnes in 1 each. Back pain as the major symptom in these patients was relieved within an average of 9.4 days after the operation. However, the patient in whom the spondylitis was caused by Candida albicans has received this new treatment twice without success. CONCLUSIONS: Evaluation of percutaneous suction aspiration with drainage shows that it is an effective treatment for early-stage pyogenic spondylitis.

Stress fracture of the medial malleolus.

It is difficult to assess the healing of the medial malleolar stress fracture. We describe a case of medial malleolar stress fracture which was monitored its healing with repeated magnetic resonance images.

Hemarthrosis of the knee and bone contusion.

We present five patients with acute traumatic hemarthrosis of the knee who also had hemorrhage within the bone marrow around the knee detected by magnetic resonance imaging. No additional bony, ligamentous, meniscal or osteochondral injuries were evident from clinical examination, initial and repeated plain radiographs, or magnetic resonance imaging. The main symptoms were sharply localized pain and tenderness of the contused area. When hemarthrosis was demonstrated at the first office visit, three cases had fat droplets in the blood. Two patients with hemarthrosis of the knee undergoing arthroscopy showed no obvious lesions of intra-articular structures. Instead they showed congestion of the joint capsule located in accordance with the traumatic episode. Magnetic resonance imaging demonstrated marrow abnormalities as diffusely low and high signal intensity areas in T1-weighted and T2-weighted images, respectively, in locations consistent with the traumatic episode and the symptoms. These disappeared within an average of 13 weeks (range in 4 cases, 12 to 16 weeks). These findings were considered diagnostic of bone contusion. Symptom duration was about 2 weeks in all cases. In conclusion the clinician should consider the possibility of coexisting bone contusion in cases of acute traumatic hemarthrosis of the knee with no evidence of bone or intra-articular lesions on clinical examination and conventional radiographs.

MRI monitoring of tarsal navicular stress fracture healing--a case report.

Stress fractures of the tarsal navicular bone are rare injury and assessing its healing is difficult. Tenderness over the tarsal navicular on physical examination is the most reliable sign for assessing fracture healing. However, it is desirable to assess fracture healing with some imaging method. We used serial magnetic resonance imaging (MRI) to assess healing in a patient with a tarsal navicular stress fracture. MRI was useful for the assessment of fracture healing of tarsal navicular stress fracture in our case.

Efficacy of aquatic exercises for patients with low-back pain.

We have studied 35 patients (25 female and 10 male) with low-back pain who were managed with aquatic exercises after an appropriate period of treatment for their condition in the medical institution. The exercises employed consisted of strengthening exercises for the abdominal, gluteal, and leg muscles, stretching of the back, hip, hamstrings, and calf muscles, walking in water, and swimming. All the patients had been participating in the exercise program for more than 6 months. The frequency of performing exercises was once a week for 7 patients, twice a week for 19, and 3 or more times a week for the remaining patients. The method used in this study was a survey questionnaire which was composed of questions about the patient's physical and psychological condition. Those patients who had performed exercises twice or more in a week showed a more significant improvement in the physical score than those who performed exercises only once a week. More than 90% of the patients felt they had improved after 6 months of participation in the program. The improvement in physical score was independent of the initial ability in swimming. The results obtained suggested that exercises in water may be one of the most useful modes of exercise for a patient with low-back pain.

A new reduction technique for a patellar fracture.

We report 4 cases of patellar fracture with skin injury over the patella which were treated operatively immediately after onset with a new reduction technique using Ilizarov pins. This technique made it possible to perform early operative treatment even in the cases with skin injury over the patella which prevents conventional surgical approach.